RNA levels of human retrovirus receptors Pit1 and Pit2 do not correlate with infectibility by three retroviral vector pseudotypes

The gibbon ape leukemia virus (GaLV) and the amphotropic murine leukemia virus (A-MuLV) infect human cells via specific receptors, Pit1 and Pit2, respectively. mRNA levels of these receptors were determined by Northern analysis and for Pit2 in addition by quantitative RT-PCR. Pit1 and Pit2 were expressed in different amounts in human tissues and cell lines; Pit1-specific mRNA was generally more abundant than Pit2 mRNA. No correlation was found between Pit1 and Pit2 RNA levels and infectibility by GaLV and A-MuLV pseudotyped vectors, respectively. GaLV and A-MuLV revealed a partial reciprocal interference. MuLV-10A1 can utilize both Pit1 and Pit2 for entry into cells but could not infect any of the 14 human cell lines more efficiently than A-MuLV or GaLV. Interference assays suggested that MuLV-10A1 has a higher affinity for and infected most cells predominantly by Pit2. However, at least in one cell line it used Pit1 more efficiently for entry. We conclude that (1) Pit1 and Pit2 mRNA levels in human cells are not indicative of the infectibility by GaLV and A-MuLV pseudotypes, respectively; (2) A-MuLV can infect target cells as efficiently as can GaLV, although Pit2 RNA is less abundant than Pit1 RNA; (3) factor(s) in addition to the presence of Pit1 and Pit2 are involved in retroviral infection; and (4) MuLV-10A1 pseudotype does not infect human cells more efficiently than do A-MuLV and GaLV pseudotypes.     

Identification of envelope protein residues required for the expanded host range of 10A1 murine leukemia virus

The 10A1 murine leukemia virus (MuLV) is a recombinant type C retrovirus isolated from a mouse infected with amphotropic MuLV (A-MuLV). 10A1 and A-MuLV have 91% amino acid identity in their envelope proteins yet display different host ranges. For example, CHO-K1 cells are resistant to A-MuLV but susceptible to infection by 10A1. We have now determined that retroviral vectors bearing altered A-MuLV envelope proteins containing 10A1-derived residues at positions 71 (A71G), 74 (Q74K), and 139 (V139M) transduce CHO-K1 cells at efficiencies similar to those achieved with 10A1 enveloped vectors. A-MuLV enveloped retroviral vectors with these three 10A1 residues were also able to transduce A-MuLV-infected NIH 3T3 cells. This observation is consistent with the ability of vectors bearing this altered A-MuLV envelope protein to recognize the 10A1-specific receptor present on NIH 3T3 cells and supports the possibility that residues at positions 71, 74, and 139 of the 10A1 envelope SU protein account for the expanded host range of 10A1.     

Simian sarcoma-associated virus fails to infect Chinese hamster cells despite the presence of functional gibbon ape leukemia virus receptors

We have sequenced the envelope genes from each of the five members of the gibbon ape leukemia virus (GALV) family of type C retroviruses. Four of the GALVs, including GALV strain SEATO (GALV-S), were originally isolated from gibbon apes, whereas the fifth member of this family, simian sarcoma-associated virus (SSAV), was isolated from a woolly monkey and shares 78% amino acid identity with GALV-S. To determine whether these viruses have identical host ranges, we evaluated the susceptibility of several cell lines to either GALV-S or SSAV infection. GALV-S and SSAV have the same host range with the exception of Chinese hamster lung E36 cells, which are susceptible to GALV-S but not SSAV. We used retroviral vectors that differ only in their envelope composition (e.g., they contain either SSAV or GALV-S envelope protein) to show that the envelope of SSAV restricts entry into E36 cells. Although unable to infect E36 cells, SSAV infects GALV-resistant murine cells expressing the E36-derived viral receptor, HaPit2. These results suggest that the receptors present on E36 cells function for SSAV. We have constructed several vectors containing GALV-S/SSAV chimeric envelope proteins to map the region of the SSAV envelope that blocks infection of E36 cells. Vectors bearing chimeric envelopes comprised of the N-terminal region of the GALV-S SU protein and the C-terminal region of SSAV infect E36 cells, whereas vectors containing the N-terminal portion of the SSAV SU protein and C-terminal portion of GALV-S fail to infect E36 cells. This finding indicates that the region of the SSAV envelope protein responsible for restricting SSAV infection of E36 cells lies within its amino-terminal region.     

A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: possible role in G protein-coupled signal transduction

Although present in many copies in the mouse genome, xenotropic murine leukemia viruses cannot infect cells from laboratory mice because of the lack of a functional cell surface receptor required for virus entry. In contrast, cells from many nonmurine species, including human cells, are fully permissive. Using an expression library approach, we isolated a cDNA from HeLa cell RNA that conferred susceptibility to xenotropic envelope protein binding and virus infection when expressed in nonpermissive cells. The deduced product is a 696-aa multiple-membrane spanning molecule, is widely expressed in human tissues, and shares homology with nematode, fly, and plant proteins of unknown function as well as with the yeast SYG1 protein, which has been shown to interact with a G protein. This molecule also acts as a receptor for polytropic murine leukemia viruses, consistent with observed interference between xenotropic and polytropic viruses in some cell types. This xenotropic and polytropic retrovirus receptor (XPR1) is the fourth identified molecule having multiple membrane spanning domains among mammalian type C oncoretrovirus receptors and may play a role in G protein-coupled signal transduction, as do the chemokine receptors required for HIV entry.     

Mutational analysis of the virus and monoclonal antibody binding sites in MHVR, the cellular receptor of the murine coronavirus mouse hepatitis virus strain A59

The primary cellular receptor for mouse hepatitis virus (MHV), a murine coronavirus, is MHVR (also referred to as Bgp1a or C-CAM), a transmembrane glycoprotein with four immunoglobulin-like domains in the murine biliary glycoprotein (Bgp) subfamily of the carcinoembryonic antigen (CEA) family. Other murine glycoproteins in the Bgp subfamily, including Bgp1b and Bgp2, also can serve as MHV receptors when transfected into MHV-resistant cells. Previous studies have shown that the 108-amino-acid N-terminal domain of MHVR is essential for virus receptor activity and is the binding site for monoclonal antibody (MAb) CC1, an antireceptor MAb that blocks MHV infection in vivo and in vitro. To further elucidate the regions of MHVR required for virus receptor activity and MAb CC1 binding, we constructed chimeras between MHVR and other members of the CEA family and tested them for MHV strain A59 (MHV-A59) receptor activity and MAb CC1 binding activity. In addition, we used site-directed mutagenesis to introduce selected amino acid changes into the N-terminal domains of MHVR and these chimeras and tested the abilities of these mutant glycoproteins to bind MAb CC1 and to function as MHV receptors. Several recombinant glycoproteins exhibited virus receptor activity but did not bind MAb CC1, indicating that the virus and MAb binding sites on the N-terminal domain of MHVR are not identical. Analysis of the recombinant glycoproteins showed that a short region of MHVR, between amino acids 34 and 52, is critical for MHV-A59 receptor activity. Additional regions of the N-terminal variable domain and the constant domains, however, greatly affected receptor activity. Thus, the molecular context in which the amino acids critical for MHV-A59 receptor activity are found profoundly influences the virus receptor activity of the glycoprotein.     

The dual-function hamster receptor for amphotropic murine leukemia virus (MuLV), 10A1 MuLV, and gibbon ape leukemia virus is a phosphate symporter

Previously, we showed that the amphotropic receptor homolog in hamster cells functions as a receptor not only for amphotropic murine leukemia viruses and 10A1 murine leukemia virus but also for gibbon ape leukemia virus (C.A. Wilson, K. B. Farrell, and M. V. Eiden, J. Virol. 68:7697-7703, 1994). Here, we demonstrate that this receptor functions as a sodium-dependent Pi transporter and that Na-Pi uptake can be specifically blocked following infection with either amphotropic murine leukemia virus, 10A1 murine leukemia virus, or gibbon ape leukemia virus.     

Requirements for different components of the host cell cytoskeleton distinguish ecotropic murine leukemia virus entry via endocytosis from entry via surface fusion

Murine ecotropic leukemia viruses use a common receptor for entry into host cells; however, the site of virus fusion appears to differ with the host cell. Entry in mouse NIH 3T3 fibroblasts is by endocytosis, whereas entry in rat XC sarcoma cells is by surface fusion. We report here the identification of a step common to both entry pathways, as well as of a step unique to the endocytic pathway. Recent demonstration of the clustering of the virus receptor on rat cells suggested a possible interaction of the receptor with the cellular cytoskeleton (M. H. Woodard, W. A. Dunn, R. O. Laine, M. Malandro, R. McMahon, O. Simell, E. R. Block, and M. S. Kilberg, Am. J. Physiol. 266:E817-E824, 1994). We tested the hypothesis that such an interaction might influence receptor function. We found that entry into NIH 3T3 and XC cells was greatly diminished by the disruption of the actin network before but not shortly after virus internalization, suggesting the actin network plays a critical role in an early step common to both entry pathways. Disruption of microtubules before and shortly after virus internalization markedly reduced entry in NIH 3T3 cells, while entry into XC cells remained efficient. These data suggest that intact microtubules are required in a postpenetration step unique to efficient virus entry via endocytosis. The physiological function of the receptor was not affected by disruption of either the actin network or the microtubules, as the uptake of cationic amino acids in NIH 3T3 and XC cells was comparable to that in control cells even when the cytoskeleton remained disrupted for as long as 3 h.     

Intrinsic human immunodeficiency virus type 1 resistance of hematopoietic stem cells despite coreceptor expression

Interactions of human immunodeficiency virus type 1 (HIV-1) with hematopoietic stem cells may define restrictions on immune reconstitution following effective antiretroviral therapy and affect stem cell gene therapy strategies for AIDS. In the present study, we demonstrated mRNA and cell surface expression of HIV-1 receptors CD4 and the chemokine receptors CCR-5 and CXCR-4 in fractionated cells representing multiple stages of hematopoietic development. Chemokine receptor function was documented in subsets of cells by calcium flux in response to a cognate ligand. Productive infection by HIV-1 via these receptors was observed with the notable exception of stem cells, in which case the presence of CD4, CXCR-4, and CCR-5, as documented by single-cell analysis for expression and function, was insufficient for infection. Neither productive infection, transgene expression, nor virus entry was detectable following exposure of stem cells to either wild-type HIV-1 or lentivirus constructs pseudotyped in HIV-1 envelopes of macrophage-tropic, T-cell-tropic, or dualtropic specificity. Successful entry into stem cells of a vesicular stomatitis virus G protein-pseudotyped HIV-1 construct demonstrated that the resistance to HIV-1 was mediated at the level of virus-cell membrane fusion and entry. These data define the hematopoietic stem cell as a sanctuary cell which is resistant to HIV-1 infection by a mechanism independent of receptor and coreceptor expression that suggests a novel means of cellular protection from HIV-1.     

Early activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase in response to binding of simian immunodeficiency virus to Jurkat T cells expressing CCR5 receptor

We have shown that the binding of simian immunodeficiency virus (SIV) to Jurkat T cells expressing CD4 receptor strongly induces mitogen-activated protein (MAP) kinase kinase (MEK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) and only weakly induces p38 MAP kinase and c-Jun N-terminal kinase (JNK). Similarly, T-tropic NL4-3 virus, which uses both CD4 and CXCR4 receptors for entry, stimulated in these cells the MEK/ERK MAP kinase (MAPK) pathway in a CD4 receptor-dependent manner (Popik and Pitha, 1998). In contrast, both macrophage-tropic SIVmac316 and T cell-tropic SIVmac239, which in addition to CD4 require CCR5 coreceptor for entry, significantly enhanced early MEK/ERK, p38 MAPK, and JNK signaling in Jurkat cells expressing constitutively or transiently the CCR5 receptor. Together, this study provides the evidence that viruses using CXCR4 or CCR5 receptors for entry may differentially use signaling properties of their specific coreceptors to stimulate MAP kinase cascades. In addition, although SIVmac239 and SIVmac316 use different structural domains of the CCR5 receptor for entry, both viruses stimulate early phosphorylation of MEK, ERK, p38, and JNK independently of their tropism and replication.     

Chemokine coreceptor usage by diverse primary isolates of human immunodeficiency virus type 1

We tested chemokine receptor subset usage by diverse, well-characterized primary viruses isolated from peripheral blood by monitoring viral replication with CCR1, CCR2b, CCR3, CCR5, and CXCR4 U87MG.CD4 transformed cell lines and STRL33/BONZO/TYMSTR and GPR15/BOB HOS.CD4 transformed cell lines. Primary viruses were isolated from 79 men with confirmed human immunodeficiency virus type 1 (HIV-1) infection from the Chicago component of the Multicenter AIDS Cohort Study at interval time points. Thirty-five additional well-characterized primary viruses representing HIV-1 group M subtypes A, B, C, D, and E and group O and three primary simian immunodeficiency virus (SIV) isolates were also used for these studies. The restricted use of the CCR5 chemokine receptor for viral entry was associated with infection by a virus having a non-syncytium-inducing phenotype and correlated with a reduced rate of disease progression and a prolonged disease-free interval. Conversely, broadening chemokine receptor usage from CCR5 to both CCR5 and CXCR4 was associated with infection by a virus having a syncytium-inducing phenotype and correlated with a faster rate of CD4 T-cell decline and progression of disease. We also observed a greater tendency for infection with a virus having a syncytium-inducing phenotype in men heterozygous for the defective CCR5 Delta32 allele (25%) than in those men homozygous for the wild-type CCR5 allele (6%) (P = 0.03). The propensity for infection with a virus having a syncytium-inducing phenotype provides a partial explanation for the rapid disease progression among some men heterozygous for the defective CCR5 Delta32 allele. Furthermore, we did not identify any primary viruses that used CCR3 as an entry cofactor, despite this CC chemokine receptor being expressed on the cell surface at a level commensurate with or higher than that observed for primary peripheral blood mononuclear cells. Whereas isolates of primary viruses of SIV also used STRL33/BONZO/TYMSTR and GPR15/BOB, no primary isolates of HIV-1 used these particular chemokine receptor-like orphan molecules as entry cofactors, suggesting a limited contribution of these other chemokine receptors to viral evolution. Thus, despite the number of chemokine receptors implicated in viral entry, CCR5 and CXCR4 are likely to be the physiologically relevant chemokine receptors used as entry cofactors in vivo by diverse strains of primary viruses isolated from blood.     

The ectodomain of a novel member of the immunoglobulin subfamily related to the poliovirus receptor has the attributes of a bona fide receptor for herpes simplex virus types 1 and 2 in human cells

We report on the functional cloning of a hitherto unknown member of the immunoglobulin (Ig) superfamily selected for its ability to confer susceptibility to herpes simplex virus (HSV) infection on a highly resistant cell line (J1.1-2 cells), derived by exposure of BHKtk- cells to a recombinant HSV-1 expressing tumor necrosis factor alpha (TNF-alpha). The sequence of herpesvirus Ig-like receptor (HIgR) predicts a transmembrane protein with an ectodomain consisting of three cysteine-bracketed domains, one V-like and two C-like. HIgR shares its ectodomain with and appears to be an alternative splice variant of the previously described protein PRR-1 (poliovirus receptor-related protein). Both HIgR and PRR-1 conferred on J1.1-2 cells susceptibility to HSV-1, HSV-2, and bovine herpesvirus 1. The viral ligand of HIgR and PRR-1 is glycoprotein D, a constituent of the virion envelope long known to mediate viral entry into cells through interaction with cellular receptor molecules. Recently, PRR-1, renamed HveC (herpesvirus entry mediator C), and the related PRR-2, renamed HveB, were reported to mediate the entry of HSV-1, HSV-2, and bovine herpesvirus 1, and the homologous poliovirus receptor was reported to mediate the entry of pseudorabies virus (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618-1620, 1998; M. S. Warner, R. J. Geraghty, W. M. Martinez, R. I. Montgomery, J. C. Whitbeck, R. Xu, R. J. Eisenberg, G. H. Cohen, and P. G. Spear, Virology 246:179-189, 1998). Here we further show that HIgR or PRR-1 proteins detected by using a monoclonal antibody to PRR-1 are widely distributed among human cell lines susceptible to HSV infection and commonly used for HSV studies. The monoclonal antibody neutralized virion infectivity in cells transfected with HIgR or PRR-1 cDNA, as well as in the human cell lines, indicating a direct interaction of virions with the receptor molecule, and preliminarily mapping this function to the ectodomain of HIgR and PRR-1. Northern blot analysis showed that HIgR or PRR-1 mRNAs were expressed in human tissues, with the highest expression being detected in nervous system samples. HIgR adds a novel member to the cluster of Ig superfamily members able to mediate the entry of alphaherpesviruses into cells. The wide distribution of HIgR or PRR-1 proteins among human cell lines susceptible to HSV infection, coupled with the neutralizing activity of the antibody in the same cells, provides direct demonstration of the actual use of this cluster of molecules as HSV-1 and HSV-2 entry receptors in human cell lines. The high level of expression in samples from nervous system makes the use of these proteins in human tissues very likely. This cluster of molecules may therefore be considered to constitute bona fide receptors for HSV-1 and HSV-2.     

Enhancement of human immunodeficiency virus type 1 infection by the CC-chemokine RANTES is independent of the mechanism of virus-cell fusion

We have studied the effects of CC-chemokines on human immunodeficiency virus type 1 (HIV-1) infection, focusing on the infectivity enhancement caused by RANTES. High RANTES concentrations increase the infectivity of HIV-1 isolates that use CXC-chemokine receptor 4 for entry. However, RANTES can have a similar enhancing effect on macrophagetropic viruses that enter via CC-chemokine receptor 5 (CCR5), despite binding to the same receptor as the virus. Furthermore, RANTES enhances the infectivity of HIV-1 pseudotyped with the envelope glycoprotein of murine leukemia virus or vesicular stomatitis virus, showing that the mechanism of enhancement is independent of the route of virus-cell fusion. The enhancing effects of RANTES are not mediated via CCR5 or other known chemokine receptors and are not mimicked by MIP-1alpha or MIP-1beta. The N-terminally modified derivative aminooxypentane RANTES (AOP-RANTES) efficiently inhibits HIV-1 infection via CCR5 but otherwise mimics RANTES by enhancing viral infectivity. There are two mechanisms of enhancement: one apparent when target cells are pretreated with RANTES (or AOP-RANTES) for several hours, and the other apparent when RANTES (or AOP-RANTES) is added during virus-cell absorption. We believe that the first mechanism is related to cellular activation by RANTES, whereas the second is an increase in virion attachment to target cells.     

Identification of a cellular receptor for subgroup E avian leukosis virus

Genetic studies in chickens and receptor interference experiments have indicated that avian leukosis virus (ALV)-E may utilize a cellular receptor related to the receptor for ALV-B and ALV-D. Recently, we cloned CAR1, a tumor necrosis factor receptor (TNFR)-related protein, that serves as a cellular receptor for ALV-B and ALV-D. To determine whether the cellular receptor for ALV-E is a CAR1-like protein, a cDNA library was made from turkey embryo fibroblasts (TEFs), which are susceptible to ALV-E infection, but not to infection by ALV-B and ALV-D. The cDNA library was screened with a radioactively labeled CAR1 cDNA probe, and clones that hybridized with the probe were isolated. A 2.3-kb cDNA clone was identified that conferred susceptibility to ALV-E infection, but not to ALV-B infection, when expressed in transfected human 293 cells. The functional cDNA clone is predicted to encode a 368 amino acid protein with significant amino acid similarity to CAR1. Like CAR1, the TEF protein is predicted to have two extracellular TNFR-like cysteine-rich domains and a putative death domain similar to those of TNFR I and Fas. Flow cytometric analysis and immunoprecipitation experiments demonstrated specific binding between the TEF CAR1-related protein and an immunoadhesin composed of the surface (SU) envelope protein of subgroup E (RAV-0) virus fused to the constant region of a rabbit immunoglobulin. These two activities of the TEF CAR1-related protein, specific binding to ALV-E SU and permitting entry only of ALV-E, have unambiguously identified this protein as a cellular receptor specific for subgroup E ALV.     

A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infection

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit human immunodeficiency virus type 1 (HIV-1) entry and infection. The CXC chemokine receptor CXCR4/fusin is a receptor for pre-B cell growth stimulating factor (PBSF)/stromal cell-derived factor 1 (SDF-1) and serves as a coreceptor for the entry of T cell line-tropic HIV-1 strains. Thus, the development of CXCR4 antagonists or agonists may be useful in the treatment of HIV-1 infection. T22 ([Tyr5,12,Lys7]-polyphemusin II) is a synthesized peptide that consists of 18 amino acid residues and an analogue of polyphemusin II isolated from the hemocyte debris of American horseshoe crabs (Limulus polyphemus). T22 was found to specifically inhibit the ability of T cell line-tropic HIV-1 to induce cell fusion and infect the cell lines transfected with CXCR4 and CD4 or peripheral blood mononuclear cells. In addition, T22 inhibited Ca2+ mobilization induced by pre-B cell growth stimulating factor (PBSF)/SDF-1 stimulation through CXCR4. Thus, T22 is a small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 entry into target cells.     

A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for Env-mediated fusion and virus infectivity

Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an alpha-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.     

Functional region IV of glycoprotein D from herpes simplex virus modulates glycoprotein binding to the herpesvirus entry mediator

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Delta290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Delta290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 x 10(-8) M versus 3.2 x 10(-6) M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Delta290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster kon rather than to a slower koff. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 x 10(-5) M) than did gD1(306t) due to a more rapid koff. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.     

Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses

Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.     

Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates

Microglia are the main human immunodeficiency virus (HIV) reservoir in the central nervous system and most likely play a major role in the development of HIV dementia (HIVD). To characterize human adult microglial chemokine receptors, we analyzed the expression and calcium signaling of CCR5, CCR3, and CXCR4 and their roles in HIV entry. Microglia expressed higher levels of CCR5 than of either CCR3 or CXCR4. Of these three chemokine receptors, only CCR5 and CXCR4 were able to transduce a signal in microglia in response to their respective ligands, MIP-1beta and SDF-1alpha, as recorded by single-cell calcium flux experiments. We also found that CCR5 is the predominant coreceptor used for infection of human adult microglia by the HIV type 1 dementia isolates HIV-1DS-br, HIV-1RC-br, and HIV-1YU-2, since the anti-CCR5 antibody 2D7 was able to dramatically inhibit microglial infection by both wild-type and single-round luciferase pseudotype reporter viruses. Anti-CCR3 (7B11) and anti-CXCR4 (12G5) antibodies had little or no effect on infection. Last, we found that virus pseudotyped with the DS-br and RC-br envelopes can infect cells transfected with CD4 in conjunction with the G-protein-coupled receptors APJ, CCR8, and GPR15, which have been previously implicated in HIV entry.     

Persistent infection promotes cross-species transmissibility of mouse hepatitis virus

Persistent infection with mouse hepatitis virus (MHV) strain A59 in murine DBT (delayed brain tumor) cells resulted in the emergence of host range variants, designated V51A and V51B, at 210 days postinfection. These host range mutants replicated efficiently in normally nonpermissive Chinese hamster ovary (CHO), in human hepatocarcinoma (HepG2), and to a lesser extent in human breast carcinoma (MCF7) cell lines. Little if any replication was noted in baby hamster kidney (BHK), green African monkey kidney (COS-7), feline kidney (CRFK), and swine testicular (ST) cell lines. By fluorescent antibody (FA) staining, persistent viruses V10B and V30B, isolated at days 38 and 119 days postinfection, also demonstrated very low levels of replication in human HepG2 cells. These data suggest that persistence may rapidly select for host range expansion of animal viruses. Pretreatment of HepG2 cells with a polyclonal antibody directed against human carcinoembryonic antigens (CEA) or with some monoclonal antibodies (Col-1, Col-4, Col-12, and Col-14) that bind human CEA significantly inhibited V51B infection. Under identical conditions, little or no blockade was evident with other monoclonal antibodies (kat4c or Col-6) which also bind the human CEA glycoproteins. In addition, an antibody (EDDA) directed against irrelevant antigens did not block V51B replication. Pretreatment with the Col-4 and Col-14 antibodies did not block Sindbis virus replication in HepG2 cells or MHV infection in DBT cells, suggesting that one or more CEA glycoproteins likely functioned as receptors for V51B entry into human cell lines. To test this hypothesis, the human biliary glycoprotein (Bgp) and CEA genes were cloned and expressed in normally nonpermissive BHK cell lines by using noncytopathic Sindbis virus replicons (pSinRep19). By growth curves and FA staining, human CEA and to a much lesser extent human Bgp functioned as receptors for V51B entry. Furthermore, V51B replication was blocked with polyclonal antiserum directed against human CEA and Bgp. Under identical conditions, the parental MHV strain A59 failed to replicate in BHK cells expressing human Bgp or CEA. These data suggest that MHV persistence may promote virus cross-species transmissibility by selecting for virus variants that recognize phylogenetic homologues of the normal receptor.